Over 8 million people in the US suffer from peripheral arterial disease (PAD), which is characterized by narrowing of the arteries that supply blood flow to the limbs, leading to tissue ischemia. A central feature of PAD is dysfunction of the vascular endothelial cells (ECs) that control vascular reactivity and angiogenesis. We previously demonstrated that ECs derived from human induced pluripotent stem cells (iPSC-ECs) can improve blood perfusion in animals with induced hindlimb ischemia, an experimental model of PAD. However, poor cell survival limited their angiogenic potential. To address this limitation, we seek to develop aligned nanofibrillar collagen scaffolds that mimic the crimped (wavy) structure of native collagen fibrils. In comparison to injectable scaffolds, which lack organized nano-scale structure and mechanical integrity, our preliminary data suggests that aligned nanofibrillar scaffolds provide structural support for guiding the organization of newly formed vessels, directing cell survival, and stimulating angiogenesis. The global hypothesis is that crimped aligned nanofibrillar scaffolds seeded with iPSC-ECs will enhance cell survival, accelerate vascular network formation, and induce angiogenesis in the ischemic limb in small and large animal PAD models. In this resubmission application, Specific Aim 1 will test the hypothesis that crimped aligned nanofibrillar scaffolds, in contrast to randomly oriented scaffolds, will enhance iPSC-EC survival and angiogenesis in vitro. For up to 14 days, cell viability and angiogenesis (migration, vascular network formation, and cytokine production) will be quantitatively compared between iPSC-ECs that are seeded on aligned or randomly oriented scaffolds under hypoxia (1% O2). To validate these findings in vivo, Specific Aim 2 will compare the temporal process of angiogenesis, arteriogenesis, and cell survival after implantation of the iPSC-EC-seeded aligned nanofibrillar scaffold into the ischemic limb. Over 28 days, cell survival, blood perfusion recovery, and formation of new microvasculature will be quantitatively assessed by bioluminescence imaging, laser Doppler blood spectroscopy, and micro computed tomography imaging, respectively, between cells seeded on aligned or randomly oriented scaffolds. Towards clinical translation, Specific Aim 3 will be an exploratory study in which iPSC-ECs seeded on aligned or randomly oriented scaffolds will be implanted into an ovine limb ischemia model. Cell survival will be tracked by. The temporal process of microvessel formation and blood perfusion recovery will be assessed by computed tomography and fluorescence assisted angiography, respectively. Anatomical tunneling of the scaffolds to the ischemic limb will also be examined as a clinically translatable minimally invasive delivery approach. Together, the results of these studies will lead to the development of biomimetic nanofibrillar scaffolds to improve the clinical efficacy of stem cell therapy to PAD patients. We have generated the preliminary scientific components and assembled the expertise to hopefully successfully achieve these goals.